Targeted recombination is a powerful tool for introducing precise modifications into mammalian genomes. Coupled with ES cell technology, gene targeting has opened the way for full scale reverse genetics in mammals. Gene targeting has enormous potential for answering fundamental questions about gene expression, development, cancer, immunity, and physiology, for creating animal models of human diseases, and for treating genetic diseases in humans. In the short time since targeting to a native mammalian gene was first demonstrated in 1985 several dozen mammalian genes have been targeted. Gene targeting in mammalian cells, however, is by no means easy, primarily because targeted events occur at a very low frequency and are obscured by a 100-10000 fold higher frequency of random integration events. The efficiency of targeting into different genes varies by more than four orders of magnitude. This application proposes to use the selectable, well characterized APRT gene in CHO cells to study the basis for targeting variability in hopes of harnessing it to improve gene targeting. Several potential influences will be studied. (1) RECOMBINOGENIC SEQUENCES. The APRT system will be used to search for recombinogenic sequences in two ways: (i) potential candidates that have been implicated in other studies will be screened; and (ii) stimulatory sequences will be directly selected. (2) DOUBLE-STRAND BREAKS. The possibility that double-strand breaks in the target gene may stimulate targeted recombination will be investigated by inserting the HO endonuclease recognition site into the APRT gene. Targeting will be in the presence and absence of the HO endonuclease. (3) TRANSCRIPTION STATUS. The promoter for the endogenous APRT gene will be replaced with a tunable promoter to measure the effects of different levels of transcription on the frequency of gene targeting. (4) RECOMBINATION GENES. A novel genetic approach based on protein-protein interactions in yeast will be used to isolate mammalian recombination genes. Collectively, these studies will advance understanding of mammalian gene targeting and may lead to improved targeting frequencies.